A major pathway used by the malaria parasite for invasion is mediated by sialic acid (SA) residues of glycophorins present on the erythrocyte surface and the erythrocyte binding antigen (EBA) family of parasite proteins. However, some P. falciparum strains have the ability to invade neuraminidase-treated erythrocytes which lack SA, demonstrating the existence of alternative SA-independent invasion mechanisms. The erythrocyte receptors that mediate SA-independent invasion had previously been unknown until we recently identified complement receptor 1 (CR1) as the major SA-independent invasion receptor used by laboratory strains of P. falciparum. Subsequent studies have implicated the P. falciparum protein reticulocyte-binding like homologue 4 (Rh4) as the ligand for CR1. This application will extend our investigations to clinical isolates of P. falciparum in Ghana to examine the molecular mechanisms used by field parasites for invasion of erythrocytes in semi-immune individuals and to determine the changes in invasion phenotypes that allow the parasites to evade the adaptive immune response. Parasite isolates collected from three geographically distinct zones of Ghana with varying transmission intensities will be examined for their erythrocyte invasion mechanisms by testing their ability to invade erythrocytes treated with neuraminidase, trypsin, or chymotrypsin, which selectively disrupt specific erythrocyte receptors. In addition, the contribution of CR1 as a receptor will be investigated by measuring the ability of anti-CR1 antibodies and soluble CR1 to inhibit erythrocyte invasion. Erythrocyte CR1 expression in semi-immune individuals will also be measured by flow cytometry and the association with peripheral parasite density will be investigated. In addition, titers of antibodies against the major SA-dependent ligands including, EBA-140, EBA-175, and EBA-181, as well as the CR1 ligand Rh4, will be determined by ELISA and their impact on the relationship between CR1 and parasitemia will be examined using a multivariate statistical model. For parasite isolates whose invasion phenotypes are determined, gene expression of the known SA-independent parasite ligands Rh2a, Rh2b, and Rh4, as well as candidate ligands that will be identified in our ongoing investigations of CR1 ligands, will be determined by quantitative real time RT-PCR. The relationship between erythrocyte receptor usage, anti-EBA antibody titers and ligand gene expression will then be examined to definitively show if increased anti-EBA titers are associated with an increased CR1 ligand gene expression and switch to CR1-dependent invasion phenotype. Successful completion of these proposed studies will substantially increase our understanding of the invasion mechanisms that are used by malaria parasites in Ghana which has broad implications on the design of potential blood- stage interventions.